Inoculation experiment
In order to investigate potential trade-offs between within-host
infection load and transmission potential, and whether coinfection with
PlLV affects P. subordinaria infection or trade-offs, we
performed a laboratory trial using two P. subordinaria strains
(P29 and P43 originating from populations 1720 and 861, respectively) ,
one PlLV strain and one P. lanceolata genotype (511-14)
originating from the Åland Islands. Altogether 32 plants were used in
the experiment. To compare P. subordinaria performance and
trade-offs alone and in coinfection with PlLV, we inoculated half of the
plants with a PlLV strain originating from population 3301 and
maintained in P. lanceolata, whereas half of the plants received
mock inoculation with phosphate buffer. Sap from PCR confirmed (Susi et
al., 2017) PlLV infected plants mixed with phosphate buffer was used for
virus inoculation. Using a syringe, one leaf per plant was inoculated
with 100 µl virus sap or phosphate buffer. After seven days from PlLV
inoculation, the plants received P. subordinaria inoculation. To
understand pathogen strain effect on trade-offs with PlLV, half of the
plants received an inoculation with strain P29, and half received an
inoculation with strain P43. The fungal strains were collected from
field, purified on sequential inoculations on oat agar plates (de Nooji
& van der Aa, 1987) and maintained on live P. lanceolata plants.
The flower stalks were inoculated by wounding the stem just below the
inflorescence with a scalpel and immediately pipetting
106 conidia/µl suspension to the wound. In the
experiment, each treatment (PILV coinfection yes/no with P.
subordinaria strain P29 or P43) was replicated eight times. The plants
were kept inside a growth chamber in 8:16 dark: light cycle at +20 °C.
To prevent possible shelf effects, the locations of plants inside the
chamber were moved daily. To quantify within-host infection load, the
size of the P. subordinaria lesion in centimetres and the length
of each flower stalk was measured once a week starting seven days post
inoculation. Lesion growth measurements were then used for calculation
of relative area under disease progress stairs (AUDPS; (Simko & Piepho,
2012)). To measure transmission potential, we observed the time and
density of pycnidia formation. Pycnidia are the fruiting bodies of the
fungus that contain the conidial spores that spread the fungus (de Nooji
& van der Aa, 1987). The time when first pycnidia were observed on each
plant was monitored. At the end of the experiment, we counted the
pycnidia density within a square centimetre on one flower stalk from
each plant using a microscope.